RNAI-Mediated Knockdown of Sugar Metabolism and Transport Genes in Bactericera Cockerelli (SULC)

Abstract

The citrus greening disease is among the most damaging disease of citrus known to be caused by an insect-transmitted, fastidious bacterial pathogen. In this study, five genes involved in sugar metabolism and transport in the gut of potato psyllid (PoP), Bactericera cockerelli (Sulc) (Psylloidea: Triozidae), a surrogate study system for the Asian citrus psyllid (ACP), were evaluated for knockdown using RNA-interference (RNAi). Knockdown was quantified by real-time quantitative PCR (qPCR) expressed as fold-change in gene expression relative to water and a non-target sequence, luciferase. Silencing AQP2 and TRET1 resulted in 20-30% PoP mortality by 9 days post-inoculation (dpi), either individually, or combined and delivered as a group, or ‘stacked’, with other dsRNA targets. Among the five targets tested for dsRNA-mediated knockdown, silencing of AGLU1 resulted in the earliest and the greatest mortality in potato psyllid, both individually and stacked. Knockdown with different combinations of the other four targets revealed that the greatest PoP mortality was achieved from knockdown with all five dsRNAs, at 65.11%. Different combinations of dsRNAs were evaluated to determine if knockdown could be attributed to additive or synergistic effects of knockdown of two or more targets. Among the dsRNAs evaluated Trehalase and Alpha-glucosidase showed synergistic effects based on the qPCR analysis and by increased mortality. Also, RNAi penetrance and persistence of knockdown were enhanced over time, by stacking certain dsRNAs. Results showed that dsRNA screening by oral ingestion of dsRNAs delivered in a 20% sucrose solution was informative for evaluating the potentially deleterious effects of either single or ‘stacked’ dsRNAs on mortality and other phenotypes.